This invention relates generally to the field of the detection of drugs and drug metabolites in biological samples. Specifically, the invention relates to the generation of antibodies that specifically bind to a metabolite of LSD, 2-oxo-3-hydroxy-LSD, or those that recognize both the metabolite and the parent drug LSD. The compositions and methods of the present invention are particularly useful for confirming the presence of LSD or LSD metabolites in a sample potentially containing interfering substances.
Although there is widespread public perception that use of LSD is no longer a societal problem, there is considerable evidence that this illicit drug continues to be used, and in some segments of the population, its use is increasing (Bonner, Drug Detection Report. 1:5 (1992)). LSD was one of the 20 controlled substances most commonly encountered in emergency rooms across the nation in 1985, reflecting continuing abuse and trafficking of this illicit drug. In the United States, seizures of LSD by the Drug Enforcement Agency doubled in 1990 over the previous year, and in England, seizures of LSD have steadily increased since mid-1988 (Microgram 23:228 (1990)). Further causes for concern are reports that LSD is particularly popular among adolescents, and in some areas, it exceeds cocaine in popularity (Seligmann, Newsweek, February 3rd, p. 66, (1992)). Factors that have contributed to the continued use of LSD are its wide availability, low cost, and the difficulty of detecting LSD use by analysis of body fluids.
Despite the long history of abuse associated with LSD, little is known concerning the disposition of LSD in humans. The lack of pharmacokinetic data on LSD is partly due to the technical difficulty of detecting and measuring the drug in physiological specimens. LSD is not considered highly toxic, although at least two cases where death was apparently a result of LSD toxicity have been reported. However, the major reason many consider LSD to be highly dangerous is that it can have serious psychological and psychotic effects which sometimes cause users to commit irrational acts resulting in injury or death. LSD is an extremely potent psychedelic drug that acts primarily on the central nervous system; only the d-isomer of the drug is pharmacologically active. Oral doses as low as 25 xcexcg can cause central nervous system disturbances such as hallucinations, distortions in sensory perception, mood changes and dream-like thought processes, as well as psychotic reactions in apparently predisposed individuals. Therefore, concentrations of LSD and LSD metabolites in blood and urine are likely to be very low. The detection of LSD in body fluids of users is especially difficult because the quantities typically ingested are very small and because the drug is rapidly and extensively converted to metabolic products. Furthermore, the drug""s low volatility, its thermal instability, and its tendency to undergo adsorptive losses during gas chromatographic analysis all contribute to the difficulty of developing a method for confirmation of LSD in body fluids.
LSD is a natural product of the rye fungus Claviceps and was first prepared synthetically in 1938. Its psychological effects were discovered following accidental ingestion. Chemically, LSD is an ergot alkaloid and, like other compounds of this class, contains lysergic acid as the basis of its structure. Structurally similar to serotonin (5-hydroxytryptamine), LSD is thought to exert its psychotomimetic effects through antagonism of serotonin activity in the brain stem. Little is known about the tissue distribution, metabolism and excretion of LSD in humans. LSD is absorbed fairly rapidly by the gastrointestinal tract, and its plasma half-life has been calculated to be about 3 hours in man. Animal studies indicate that LSD is inactivated via hepatic oxidation. It is extensively metabolized with only negligible amounts of unchanged drug appearing in the urine and feces, with most of the metabolites being excreted in the urine. Possible metabolic transformations may be hydrolysis to lysergic acid, N-demethylation to nor-LSD and oxidation to 2-oxo-LSD. Studies with urine samples from human volunteers receiving LSD demonstrate that the drug or its closely related metabolites can be detected in the urine by radioimmunoassay (RIA) for several days following administration.
Although continued illicit use of LSD has stimulated efforts to develop effective analytical methods for the detection of the drug and its metabolites in body fluids from suspected LSD users, the methods currently available are complicated, time-consuming, expensive to perform and plagued by other problems. These methods include high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS) and radioimmunoassay. One problem faced by laboratories involved in the determination of LSD is the strong tendency for LSD and derivatized LSD to undergo adsorptive losses when subjected to gas chromatography. This behavior often prevents detection of the drug at the sub-nanogram/milliliter concentrations normally encountered in body fluids from LSD users.
Commercial RIAs for LSD are available from several sources, including ABUSCREEN LSD assay ((copyright) Roche Diagnostics Systems, Nutley, N.J.) and COAT-A-COUNT LSD assay ((copyright) Diagnostic Products Corp., Los Angeles, Calif.), and these products serve as a useful and relatively inexpensive method of screening for the presence of the drug. However, RIAs are not totally specific for LSD, so that an RIA-positive specimen still has to be confirmed by a second and more specific assay if the results of the analysis could have punitive consequences. The manufacturers"" recommended cut-off concentration for considering a sample positive for LSD is 0.5 ng/ml, although lower cut-offs have been used in investigations where legal consequences were not a concern. The actual concentration of LSD in RIA-positive urine specimens is generally lower than that indicated by the RIA, and often considerably lower. Presumably the higher concentrations indicated by RIA are due to the cross-reactivity of LSD metabolites to the RIA antisera, but this conclusion cannot be substantiated until the major LSD metabolites in urine have been identified and their cross-reactivities determined.
In testing for other drugs of abuse, immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as xcex2-galactosidase and peroxidase, fluorescent molecules such as fluorescent compounds, and radioactive compounds such as 125I are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
One type of competitive binding immunoassay is based upon the reassociation of enzymatically inactive polypeptide fragments to form active enzyme as a step of generating a detectable signal utilized to determine the amount of analyte present in a sample. This type of assay, known as cloned enzyme donor immunoassay (CEDIA), is described in U.S. Pat. No. 4,708,929. In particular, a xcex2-galactosidase enzyme donor polypeptide combines with a xcex2-galactosidase enzyme acceptor polypeptide to form active xcex2-galactosidase enzyme. Conjugating a hapten, or a small analyte or an analyte analog, to the enzyme donor polypeptide at certain sites does not affect the ability to form active xcex2-galactosidase by a complementation reaction and hence does not affect the rate of xcex2-galactosidase activity when in the presence of a substrate for xcex2-galactosidase. However, when the enzyme donor-hapten conjugate is bound by anti-analyte antibody, the complementation rate is impeded, and thereby the enzyme-catalyzed reaction rate during the initial phase of the reaction is reduced. This reduction in enzyme-catalyzed reaction rate can be monitored and has been used successfully to determine a plurality of analytes using the principle of competitive inhibition whereby enzyme donor-analyte conjugate present in a reaction mixture and analyte present in a sample compete for anti-analyte antibody prior to the addition of enzyme acceptor. The complementation rate of xcex2-galactosidase formation, and hence enzyme-catalyzed reaction rate, is increased as the amount of analyte present in the sample is increased.
The preparation of antibodies to LSD for use in immunoassays to determine the drug has been accomplished by several different approaches. One approach has been to couple the carboxyl group of lysergic acid directly to an immunogenic carrier protein, i.e. poly(L-lysine) or human serum albumin using carbodiimides. See Van Vunakis, Proc. Nat. Acad. Sci., 68:1483-87 (1971); Loeffler, J. Pharm. Sci. 62:1817-20 (1973); and Voss, Psychopharmacologia 26:140-45 (1972). This approach was used in developing early RIA methods for LSD determination, but the antibodies that were produced were characterized by poor specificity for LSD and high cross-reactivities with other ergot alkaloids.
A second approach has been to couple LSD to an immunogenic carrier protein via one of the ethyl side chains at the 8-position (Ratcliffe, Clin. Chem. 23:169-74 (1977)). In another approach, bis-diazo benzidine was used to couple the carrier proteins via an aromatic substitution (Luderer, Bull. New Jersey Acad. Sci. 19:8-10 (1974)).
Finally, LSD has been coupled to an immunogenic carrier protein using a reaction between LSD, formaldehyde and bovine serum albumin. See Castro, Res. Commun. Chem. Pathol. Pharmacol. 6:879-86 (1973); Taunton-Rigby, Science 181:165-6 (1973); and Ratcliffe, Clin. Chem. 23:169-74 (1977); see also Orchin, The Vocabulary of Organic Chemistry, John Wiley and Sons, NY, p. 385 and p. 501, Figure 13.790; Furniss, Vogel""s Textbook of Practical Organic Chemistry, 4th Ed., Longman Scientific and Technical and John Wiley and Sons, NY, p. 813 (1978); and Mundy, Name Reactions and Reagents in Organic Synthesis, John Wiley and Sons, NY, p.137 (1988). The reaction product is not well-defined.
More recently, Salamone, S. J. et al. in Bioconjugate Chem 8: 896-905 (1997) reported the synthesis of an array of LSD immunogens by conjugating LSD analogs derivatized through the indole nitrogen (N-1) or N-6 position to a carrier. The antibodies generated by these immunogens exhibit broad reactivity toward LSD and several LSD metabolites. Whereas the antibodies react strongly to LSD, the antibodies have low cross-reactivity (in the range of only 30-45%, molar ratio) to several LSD metabolites, including 2PATENT -oxo-3-hydroxy-LSD. While Salamone, S. J. et al. failed to recognize that 2-oxo-3-hydroxy-LSD is indeed an endogenous LSD metabolite as oppose to be a xe2x80x9ctentativexe2x80x9d metabolite, it is now becoming evident that 2-oxo-3-hydroxy-LSD may be the most prevalent metabolite of LSD. Thus, there remains a considerable need for compositions and methods applicable for generating antibodies specific for the LSD metabolite, 2-oxo-3-hydroxy-LSD. The production of these antibodies would greatly facilitate detecting the presence of LSD or LSD metabolites in a clinical sample, and confirming LSD abuse in a clinical setting.
The present invention provides compositions and methods applicable for generating antibodies specific for a LSD metabolite, 2-oxo-3-hydroxy-LSD, or its derivatives. This invention also provides the uses of these antibodies for the detection or measurement of LSD or 2-oxo-3-hydroxy-LSD in samples obtained from subjects who may have been exposed to LSD. In various embodiments, the system allows for detection of both the parent substance and natural metabolites as they may be formed within the subject or secreted into a biological fluid, particularly urine. The sensitivity and specificity of the reagents may be used in diagnostic-grade immunoassays for screening of drugs of abuse in a clinical setting.
In one embodiment, the present invention provides novel hapten derivatives of the formulas 
wherein X is xe2x80x94L1xe2x80x94Z, where L1 is a linker containing at least one carbon atom; wherein Z is selected from the group consisting of the moieties
xe2x80x94NH2,
xe2x80x94COOH,
xe2x80x94SH, 
xe2x80x94NHxe2x80x94C(xe2x95x90O)xe2x80x94L2xe2x80x94M, 
a moiety which reacts with a protein to form a covalent bond, or any combination or repetition of the aformentioned moieties; where L2 is a linker containing at least one carbon atom; where M is halide or maleimide; and wherein J is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94 or xe2x80x94CH2xe2x80x94. L1 and L2 are preferably independently selected from the group consisting of C1-C20 hydrocarbon chains, containing zero to ten heteroatoms selected from the group consisting of N, O, and S.
In another embodiment, the present invention provides novel hapten derivatives of the formulas 
where Q is xe2x80x94L1xe2x80x94G, L1 is a linker containing at least one carbon atom, and G is selected from the group consisting of fluorescent, chemiluminescent, phosphorescent, and chromophoric compounds, a fluorescence quenching group, a radioisotopically labeled group, an electrochemically active group, an electrochemiluminescent group, a group that undergoes a change in fluorescence, phosphorescence, chemiluminescence or electrochemical property upon binding, peptides, proteins, protein fragments, immunogenic carriers, enzymes, enzyme donors, enzyme inhibitors, enzyme substrates, enzyme cofactors, enzyme prosthetic groups, solid particles, gold particles, antibodies, and nucleic acids. L1 is preferably selected from the group consisting of C1-C20 hydrocarbon chains, containing zero to ten heteroatoms selected from the group consisting of N, O, and S.
In another embodiment, the present invention provides a method of generating an antibody specific for a novel LSD metabolite, 2-oxo-3-hydroxy-LSD, or its derivatives. The method involves preparing 2-oxo-3-hydroxy-LSD immunogens having the formulas shown above, and immunizing an appropriate host to elicit an immunogen-specific immune response.
In yet another embodiment, the invention provides an antibody that binds specifically to 2-oxo-3-hydroxy-LSD or a derivative thereof. The antibody can be specific for the LSD metabolite, 2-oxo-3-hydroxy-LSD (as distinct from the parent drug LSD), or it can be specific for both the parent drug LSD and the metabolite. The antibody can be polyclonal or monoclonal, non-conjugated or conjugated to a detectable label.
In a separate embodiment, the invention provides systems that permit detection of 2-oxo-3-hydroxy-LSD, either alone or in combination with LSD itself. Testing for 2-oxo-3-hydroxy-LSD permits the practitioner to detect possible use of LSD by the subject over a longer period of time than by testing for LSD alone. Specifically, the invention includes a reagent system for use in an immunoassay, comprising an antibody of this invention along with a labeled competitive binding compound (typically an LSD derivative, or a derivative of 2-oxo-3-hydroxy-LSD) that competes with the substance being tested for binding to the antibody. The reagent system can also optionally include additional components useful in conducting an assay, such as reagents for developing a detectable signal from the labeled compound following the competition reaction, buffers, standards, or written instructions. Different reagents can be premixed in any workable combination.
Also embodied in the present invention is an immunoassay method for determining the possible presence of 2-oxo-3-hydroxy-LSD in a sample. Such method involves preparing a reaction mixture comprising the sample, an antibody of this invention, and a labeled competitive binding compound capable of competing with 2-oxo-3-hydroxy-LSD. The amount of label bound to the antibody in the reaction mixture is then determined by a suitable detection technique, such as separating the complexes and measuring the label, or measuring an effect on the labeled compound as a result of being bound to antibody, such as a change in fluorescence or enzyme activity. The amount can be correlated with exposure of the subject to LSD, and if appropriate, further steps can be taken to distinguish between LSD and the metabolite, 2-oxo-3-hydroxy-LSD. This method can be employed to confirm previous exposure to LSD over a longer period of time than is possible by testing for LSD alone. Featured reagents for conducting such immunoassay include the following: competitive binding compounds in which the label is conjugated to the LSD derivative or derivatives of 2-oxo-3-hydroxy-LSD; antibodies that are specific for 2-oxo-3-hydroxy-LSD or specific for both LSD and the metabolite; CEDIA(copyright) assay system; and LSD or 2-oxo-3-hydroxy-LSD conjugated to enzyme donors of xcex2-galactosidase.
Further provided by the present invention are assay methods for confirming the presence of the analyte, 2-oxo-3-hydroxy-LSD, and distinguishing it from interfering substances potentially present in a test sample. Samples giving a positive reaction in a direct immunoassay test are treated with a neutralizing antibody that inhibits reactivity of the true analyte, but not the interfering substance. Thus, samples giving a positive reaction in the direct test but decreased reaction in the confirmation test are marked as containing the true analyte. Samples giving a positive reaction of roughly equivalent magnitude in both the direct and confirmation test are marked as containing an interfering substance. In one embodiment, a direct assay is conducted to determine the amount of analyte and/or interfering substance in the sample. The same sample or a duplicate is treated with a neutralizing antibody in an amount sufficient to remove the analyte but not the potential interfering substance, and an assay is conducted on the treated sample. The amount detected is then compared between the treated and untreated sample. In another embodiment, the assay proceeds by preparing a reaction mixture that comprises the test sample, a detecting antibody, and a competitive binding compound, wherein the detecting antibody binds a hapten derivative in a manner that is specifically inhibitable by the analyte, 2-oxo-3-hydroxy-LSD. The amount of the detecting antibody bound to the competitive binding compound is measured. To conduct the confirmation part of the test, the same sample is treated with a neutralizing antibody, or else a duplicate sample is treated with a neutralizing antibody before, during or after the direct test. The neutralizing antibody prevents the analyte but not all the interfering substance in the sample from being available to bind the detecting antibody when an assay is conducted on the treated sample. The results from the direct and the confirmatory test are then compared. The presence of the true analyte, 2-oxo-3-hydroxy-LSD, is confirmed if there is a significant effect on the result due to use of the neutralizing antibody. Featured types of confirmatory assays of this invention include bidirectional antibody type confirmatory assays and adsorption type confirmatory assays.
Kits employed for conducting the confirmatory assays comprise a detecting antibody for the analyte, 2-oxo-3-hydroxy-LSD, and a neutralizing antibody for the analyte. The neutralizing antibody preferentially binds the analyte in comparison with the interfering substance. The neutralizing antibody is preferably either aliquoted in an amount sufficient to remove the analyte but not all the interfering substance from the sample, or a written indication is provided as to the amount required. The set of reagents also typically comprises a competitive binding compound, with the property that the detecting antibody binds the hapten derivative in a manner that is specifically inhibitable by the analyte and the interfering substance. An exemplary competitive binding compound is a hapten derivative, such as a hapten-protein conjugate or a hapten labeled with a radioisotope or fluorochrome.
Further aspects of the invention and desirable characteristics of the reagents and assay methods will be apparent from the description that follows and the appended claims.